Genomic Alterations in Advanced Breast Cancer: Role of Next generation sequencing (AKT pathway)Insights from Kuwaiti Statistics and Clinical Implications

Genomic Alterations in Advanced Breast Cancer: Role of Next generation sequencing (AKT pathway)
Insights from Kuwaiti Statistics and Clinical Implications
The PI3K/AKT/PTEN signaling pathway plays a significant role in regulating cell proliferation and survival. Activating mutations of the AKT1, PIK3CA genes, and loss-of-function alterations of the PTEN gene are associated with tumor growth and treatment resistance
NGS testing provide prognostic and predictive insights, and determine treatment eligibility for (PIK3CA/AKT inhibitors) following progression on or detecting resistance ESR1 mutation after endocrine therapy and germline BRCA
Guidelines recommend NGS testing for patients with locally advanced and metastatic HR+/HER2- breast cancer, All patients in Kuwait can receive NGS testing in KCCC

1. [email protected] Genomic Alterations in Advanced Breast Cancer: Role of Next generation sequencing (AKT pathway) Dr : Amir Abdelazim, MD Biochemical and molecular pathology 25-09-2025 Insights from Kuwaiti Statistics and Clinical Implications

2. 30 years of BRCA BRCA Genes Dr. Mary-Claire King · BRCA1 gene discovered in 1994 · BRCA2 gene discovered in 1995 first cancer susceptibility genes to be identified as increasing the risk for breast and ovarian cancers.

3. Define the role of NGS in biomarker discovery Explain genomic heterogeneity in breast cancer &PIK3CA, AKT1 and PTEN-alterations and pathway. Discuss clinical applications of liquid biopsy in breast cancer. Content-objective Review guideline recommendations for biomarker testing Kuwait experiences and Statistics

4. Breast cancer is the most common cancer and one of the leading causes of cancer-related death worldwide. 1 HR- positive breast cancer (expressing estrogen or progesterone receptors, or both), is the most common subtype, with more than 65% of tumors considered HR- positive and HER2- low or HER2-negative. 2 Collectively, mutations in PIK3CA and AKT1 and alterations in PTEN occur frequently, affecting up to 50% of patients with advanced HR- positive breast cancer. 3- 5 Endocrine therapies are widely used in this setting, but many patients develop resistance to first- line cyclin- dependent kinase (CDK) 4/6 inhibitors and estrogen receptor- targeting therapies, underscoring the need for additional endocrine therapy- based options. 6 Sung H, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 10.3322/caac.21660. National Cancer Institute. Surveillance, Epidemiology and End Results Program. Available at: https://seer.cancer.gov/statfacts/html/breast-subtypes.html. Accessed August 2024. Howell S J, et al (FAKTION). J Clin Oncol. 2022; 23:851- 64. Hortobagyi G N, et al. Correlative Analysis of Genetic Alterations and Everolimus Benefit in Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results From BOLERO- 2. J Clin Oncol. 2016; 34:419- 26. Millis S Z, et al. Landscape of phosphatidylinositol-3-kinase pathway alterations across 19784 diverse solid tumors. JAMA Oncol. 2016;2(12):1565- 73. Lin M, et al. Comparative Overall Survival of CDK4/6 Inhibitors Plus Endocrine Therapy vs. Endocrine Therapy Alone for Hormone receptor-positive, HER2-negative metastatic breast cancer. J Cancer. 2020; 10.7150/jca.48944. Introduction

5. What ? We have in KCCC lab Advantages and challenges of clinical NGS Biomarker in Breast Cancer Timeline of actionable biomarkers in HR+/HER2- Define the role of NGS in biomarker discovery

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10. The NCCN Guidelines The ESMO Guidelines Negatively impact survival outcomes Treatment Resistance Guidelines ® recommendations

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15. 1 Recommend biomarker testing for this patient population to identify actionable markers, including PIK3CA, AKT1 and PTEN alterations1−4 ASCO, ESMO and NCCN guidelines 2 preferred biomarker testing method as it can provide a detailed analysis of multiple alterations simultaneously, potentially saving tissue, time and costs vs. PCR610 NGS is the AKT1 =serine/threonine protein kinase 1; BC=breast cancer; ASCO=American Society of Clinical Oncology; ctDNA =circulating tumour DNA; ESMO=European Society for Medical Oncology; FFPE=formalin-fixed paraffin-embedded; HER2=human epidermal growth factor receptor 2; HR=hormone receptor; mBC =metastatic breast cancer; NCCN=National Comprehensive Cancer Network; NGS=next-generation sequencing; PCR=polymerase chain reaction; PIK3CA =phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PTEN =phosphatase and tensin homolog. 1. Mosele MF, et al. Ann Oncol . 2024;35:588 –606 ; 2. Henry NL, et al. J Clin Oncol. 2022;40:3205–3221; 3. Burstein HJ, et al. J Clin Oncol. 2024;42:1450–1453; 4. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines ® ). Breast Cancer. Version 4.2024. July 3, 2024. Available at: https://www.nccn.org/professionals/physician_gls/pdf/breast.pdf (Accessed September 2024); 5. Wales Cancer Network. PIK3CA -mutated breast cancer clinical management. September 2022. Available at: https://executive.nhs.wales/our-functions/networks-and-planning/cancer/wcn-documents/mutated-breast-cancer-clinical-guidance-document/ (Accessed September 2024) ; 6. Li MM, et al . J Mol Diag . 2017;19:4–23; 7. Colomer R, et al. EClinicalMedicine . 2020;25:100487; 8. Horak P, et al. ESMO Open. 2016;1:e000094; 9. Ewalt MD, et al. JAMA Oncol. 2019;5:1076; 10. Yska HAF, et al. J Clin Immunol . 2019;39:577–591; 11. Horak P, et al. ESMO Open. 2016;1:e000094; 12. Schwartzberg L, et al. Am Soc Clin Oncol. 2017;37:160–169; 13. Schmid S, et al . ESMO Open. 2022;7:100570; 14. Mateo J, et al. Ann Oncol . 2018;29:1895–1902.

16. Overview of HR+/HER2- Breast Cancer Studies that have evaluated PIK3CA Studies that have evaluated AKT1 & PTEN ethnicity and geography was shown to impact PIK3CA Genomic heterogeneity in breast cancer &PIK3CA, AKT1 and PTEN-alterations

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21. PI3K/AKT signaling pathway PIK3CA/AKT1/PTEN alterations Pathway diagram Alterations in PIK3CA, AKT1, and/or PTEN table The PI3K/AKT component and mechanism of the pathway

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26. Kuwait experiences and Statistics

27. What we have in KCCC lab for BC Somatic/ germline?

28. What ? We have in KCCC lab. Panel includes genes in the homologous recombination repair (HRR) pathway, including ATM, BRCA1, BRCA2, CDK12, CHEK1, CHEK2, PALB2, RAD51B, RAD51C , and  RAD51D

29. Kuwaiti Statistics Total cases :89 done by breast cancer tissue Jan2022-SEPT2025 we did not received tissue for breast cancer regularly so , the statistic for this panel will not be informed .

30. What we have in KCCC lab for BC • Low limit of detection0.1% 52 genes Somatic/ germline? Somatic

31. What ? We have in KCCC lab. • Low limit of detection—variant detection down to 0.1% 52 genes ctDNA are fragments of DNA that are released into the blood from apoptotic tumor cells ctDNA may also have utility in the detection of minimal residual disease and may help improve diagnostics and prognostication Tissue testing seems likely to remain first-line and gold standard

32. PI3K Inhibitors AKT Inhibitors Gain-of-Function PI3K Mutations PIK3CA is one of the most frequently mutated genes in BC, occurring in approximately 40% of HR+, HER2– ABCs Gain

33. AKT Inhibitors Gain Loss PTEN -loss PIK3CA-Gain

34. AKT Inhibitors AKT1-Gain

35. Gain Gain AKT Inhibitors

36. ESR1  encodes the  estrogen receptor alpha (ER α) Predicts Endocrine Therapy Resistance Switch to  SERDs  (e.g., fulvestrant ) or  SERD-CDK4/6 inhibitor combinations  (e.g., elacestrant ) correlates with  aggressive disease  

37. ?

38. L ow concordance for detecting PTEN biallelic deletions has been found in tumour and ctDNA samples 1,2 Mosele MF, et al. Ann Oncol . 2024;35:588 –606; 2. Sturgill E. Cancer Genomics . 2021;5:1297–131141;61; 3. Roche website. Available at: https://sequencing.roche.com/us/en/products/group/avenio-tumor-tissue-cgp-kit.html. Accessed September 2024. 4. NCCN Clinical Practice Guidelines in Oncology. Breast Cancer. v2.2024; 5. Henry NL, et al. J Clin Oncol . 2022;40:3205–3221; 6. Gennari A, et al. Ann Oncol . 2021;32(12):1475–1495; 7. Vasan N, et al. J Clin Oncol . 2024;42(16 suppl ):e15033; 8. Zaikova E, et al. NPJ Breast Cancer . 2024;10:3; 9. Xu B, et al. J Oncol . 2020:2020:4259293.. 1. Tsai C, et al. Presented at ESMO Annual Congress 2024. 13–17 September. Barcelona, Spain. Poster 418P; 2. Vasan N, et al. J Clin Oncol . 2024;42(16 suppl ):e15033 . Additional data generation is needed to support use of ctDNA testing to detect PTEN alterations. In cases where liquid biopsy results are negative for PIK3CA/AKT1/PTEN-alterations, confirmatory tissue testing should be carried out Concordance between liquid and tissue NGS (retrospective analysis) 2 ctDNA TF subgroups PIK3CA short variants AKT1 short variants PTEN short variants PTEN biallelic deletions Positive percent agreement (%) ESMO Precision Medicine Working Group updated guidance recommend tumour NGS testing for AKT1 and PTEN 1 Guidelines (NCCN, ASCO, ESMO) identify ctDNA as a viable option for specific biomarker testing in the metastatic setting ( ESR1 m, PIK3CA m 4–6 ) Detection of PTEN alterations may be poor in this sample type 7–9 Nevertheless, guidelines recommend confirmatory tissue testing if liquid biopsy results are negative , as a small number of additional patients with PIK3CA alterations may be detected 4,5 Tissue – ideally from recent metastatic tumour biopsy 1,2 If not available, archival tissue from primary tumour resection/biopsy may be tested 1 Liquid ( ctDNA ) – may be used, but is associated with a higher risk of false negatives vs. tissue (therefore repeat [tissue] testing is recommended if no mutations are detected) 1–3 One study showed concordance between tissue and liquid testing for PIK3CA/AKT1/PTEN alterations (56% vs 44%, respectively) 4

39. Kuwaiti Statistics Total cases :339 done by Liquid biopsy AKT1 11 3.2% PIK3CA 118 34.80% ESR1 152 44.80% PTEN 5 1.47% 118 152 45 Oct2023-SEPT2025

40. What we have in KCCC lab for BC Inherited Cancer panel 134 germline genes • Low limit of detection0.1% 52 genes Somatic/ germline? Somatic germline

41. Inherited Cancer panel AIP, AKT1, ALK, ANTXR1, APC, ASCC1, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, BUB1, BUB1B, CACNA1D, CBL, CDC73, CDH1, CDK4, CDKN1B, CDKN2A, CHEK2, CTHRC1, CYLD, DDB2, DICER1, EGFR, ELAC2, EPCAM, ERCC2, ERCC3, ERCC4, ERCC5, EXT1, EXT2, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FH, FLCN, GPC3, HNF1A, HNF1B HOXB13, KDR, KIT, KLLN, LIG4, MAX, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH6, MSR1, MTAP, MUTYH, MYH8, NBN, NCOA4, NF1, NF2, NTRK1, PALB2, PALLD, PDGFRB, PHOX2B, PIK3CA, PMS2, POLD1, POLE, POLH, PPM1D, PRF1, PRKAR1A, PRSS1, PTCH1, PTCH2, PTEN, PTPN11, RAD50, RAD51C, RAD51D, RB1, RECQL4, RET, RHBDF2, RNASEL, RNF168, RSPO1, RUNX1, SETBP1, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SH2D1A, SLX4, SMAD4, SMARCB1, SMARCE1, SPINK1, SPRED1, STK11, SUFU, TERT, TGFBR1, TMEM127, TP53, TSC1, TSC2, VHL, WAS, WRN, WT1, XPA, XPC, and XRCC2 134 germline genes Patient consent needed family history needed

42. Inherited Cancer panel confer a  45–80% lifetime risk of breast cancer  and a  40–60% risk of ovarian cancer BRCA1 loss disrupts  homologous recombination repair (HRR) , leading to defective DNA repair and chromosomal instability. PARP Inhibitors (e.g., Olaparib, Talazoparib ) It is estimated that 2–9% of patients with breast cancer have a gBRCAm Patients with gBRCAm have higher recurrence scores than those without

43. Kuwaiti Statistics Total breast cancer cases :2305 done by Inherited cancer panel BRCA1/2 5.2% Jan2022-SEPT2025 2022+2023 2024 2025/sept BRCA1 26 21 20 67 BRCA2 15 22 15 52 PALB2 14 ATM 13 CHEK2 17 BRIP1 6 CDH1 3 BARD1 5 RAD51D 4 EPCAM 2 PTEN 2 RAD50 2 TP53 8 RAD51C 3 Pathogenic/ Likely pathogenic Note: Variant of uncertain significance kept under follow up and when the classification change , >>>>> re-reporting Note: This statistic include only diagnosed breast cancer cases ,BRCA detected in other members of family not included

44. ClinVar database

45. NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way. aBC, advanced breast cancer; AKT, serine/threonine protein kinase; AKT1 , serine/threonine protein kinase 1; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; mBC, metastatic breast cancer; MDT, multidisciplinary team; NCCN, National Comprehensive Cancer Network; NGS, next-generation sequencing; PI3K, phosphoinositide 3-kinase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PTEN , phosphatase and tensin homolog. 1 . Li H, Prever L, Hirsch E, et al. Cancers (Basel) . 2021;13(14):3517. 2. Paplomata E, O’Regan R. Ther Adv Med Oncol. 2014;6(4):154-166. 3. Dong C, Wu J, Chen Y, et al. Front Pharmacol . 2021;12:628690. 4. Martorana F, Motta G, Pavone G, et al . Front Pharmacol . 2021;12:662232. 5. Miricescu D, Totan A, Stanescu- Spinu II, et al Int J Mol Sci . 2020;22(1):173. 6. Foundation Medicine. FoundationOne ® CDx Technical Information. Accessed May 02, 2024. https://assets.ctfassets.net/w98cd481qyp0/41rJj28gFwtxCwHQxopaEb/277379f0a3d94a6f15b7d9633a876ab4/FoundationOne_CDx_Technical_Information__FDA_Label_.pdf . 7. Vanderwalde AM, Ma E, Yu E, et al . J Clin Oncol . 2021;39(28)_ suppl , 288-288. 8. Li MM, Datto M, Duncavage EJ, et al . J Mol Diagn . 2017;19(1):4-23. 9. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines ® ) for Breast Cancer V.2.2024. © National Comprehensive Cancer Network, Inc. 2024. All rights reserved. Accessed March 26, 2024. To view the most recent and complete version of the guideline, go online to NCCN.org. 10 . Henry NL, Somerfield MR, Dayao Z , et al. J Clin Oncol. 2022;40(27):3205-3221. 11 . Gonzalez D, Mateo J, Stenzinger A, et al . J Pathol Clin Res. 2021;7(4):311-325. Conclusion The PI3K/AKT/PTEN signaling pathway plays a significant role in regulating cell proliferation and survival. Activating mutations of the AKT1, PIK3CA genes, and loss-of-function alterations of the PTEN gene are associated with tumor growth and treatment resistance NGS testing provide prognostic and predictive insights, and determine treatment eligibility for (PIK3CA/AKT inhibitors) following progression on or detecting resistance ESR1 mutation after endocrine therapy and germline BRCA Guidelines recommend NGS testing for patients with locally advanced and metastatic HR+/HER2- breast cancer, All patients in Kuwait can receive NGS testing in KCCC Ensure PIK3CA, AKT1, and/or PTEN alteration status is identified for all HR+/HER2- locally aBC or mBC cases at diagnosis or following 1L progression

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